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    The Proteome of Delta Protein Kinase C in Breast Cancer

    Scientific Abstract:
    TITLE OF PROJECT: The Proteome of Delta Protein Kianse C in Breast Cancer BACKGROUND: Protein kinase C (PKC) is a family of phospholipids-dependent serine/threonine kinases of 12 related enzymes. PKC and tumor promotions have been strongly linked because conventional and novel PKCs are activated by the tumor promoter, phorbol 12-myristate 13-acetate (PMA). Relevant to this study, elevated PKC levels have been reported in breast tumors as compared to normal breast tissue from the same patient. Using estrogen receptor (ER)-sensitive breast cancer derived cell line, MCF-7, delta PKC mediates estrogen-, FGF-2- and PMA-stimulation of Ras/ERK pathway and controls the proliferation and metastasis of tumor. Delta PKC is also involved in programmed cell death, or apoptosis. However, the direct substrates of delta PKC in this and other cellular events are not known. HYPOTHESIS: It appears that delta PKC contributes both to promotion of cell growth and to inhibition of apoptosis. I hypothesize the select substrates of activated delta PKC mediate each of these responses in breast cancer. To identify these delta PKC substrates, current screening methods using delta PKC as bait are not suitable, because they are designed to identify proteins with tight protein-protein interactions. The interaction of any kinase with its substrate is short lived and with a quick turn over. Therefore, there is a need to design screening methods to identify the interactions with such high speed turnovers. Here I propose a new method to identify specific substrates of delta PKC by utilizing delta PKC isozyme specific activator/inhibitor developed in our lab and a delta PKC-chimera that catalytically labels proteins that come in contact with the kinase with a mutant ubiquitin tag. Using this unbiased but selective tagging method, the molecular pathways downstream of delta PKC that contribute to the phenotype of breast cancer cells will be elucidated. SPECIFIC AIM: The goal of this research is to elucidate the downstream signaling pathway of delta PKC using novel isozyme-specific inhibitors and activators that are available only in the Mochly-Rosen lab. By using the ubiquitin tag method, substrate proteins of delta PKC phosphorylated during MCF-7 growth and during response to pro-apoptotic stimuli should be readily identified. STUDY DESIGN: I plan to use molecular tagging of substrates that only transiently interact with delta PKC in MCF7 cells, an estrogen receptor sensitive breast cancer derived cell line. I propose to 'hijack' a mechanism that the cell uses to label proteins in an irreversible way with a tag (called ubiquitin). Proteins labeled with ubiquitin are destined for degradation. I plan to use an altered tag that the degradation machinery does not recognize. I will devise a chimera molecule where the kinase is fused with a tagging enzyme (called ubiquitin ligase). This chimera will be able to tag any protein that interacts with our kinase. The tagged proteins can then be purified on affinity columns and identified by sensitive methods, such as mass spectroscopy. To confirm that a protein is a substrate of delta PKC, I will stimulate MCF-7 cells with delta PKC-selective agonist peptide with and without 1nM PMA. Proteins that are substrates of delta PKC will show a delta PKC-activator-dependent leftward mobility shift indicative of phosphorylation on 2-D gel analysis. These findings should be followed by studies that examine the consequences of each phosphoryaltion event on cells function POTENTHIAL OUTOCOMES: Personalized medicine using DNA chip, for example, has already helped selecting the optimal anti-tumor agents for individual patient. However, new anti-tumor agents will be elucidated if we obtain a better understanding of the molecular events in different tumor cells. When all the substrate proteins that interact with delta PKC will be identified, we will be able to design specific inhibitor of for these interactions by mimicking the interaction sites of delta PKC and each substrate protein. If the substrate protein have role in tumor promotion, the newly produced peptide inhibitor of the substrate protein may provide a basis a new anti-cancer reagent. Moreover, the methods developed in this study could be applied for the other protein kinases and phosphatases related to tumor progression.

    Lay Abstract:
    Title of Project:The Proteome of Delta Protein Kianse C in Breast Cancer INTRODUCTION TO THE RESEARCH TOPICS: Tumor behavior and characteristics are different in each patient. Therefore, the treatment should also be tailored specifically for each patient. The classification of cancer types and designing the corresponding treatment will improve patient's outcome. With the completion of The Human Genome Project, improved diagnosis of and tailoring the treatment for cancer is under development by many researchers. This application is aimed at editing the genome information with information on the proteins that these genes encode. The ultimate goal of such a project is to develop new treatments of breast cancer by understanding how the protein networks communicate and govern the responses of breast cancer cells to stimulation that promote growth and to those that promote death of the cancer cells. THE QUESTIONS AND CENTRAL HYPOTHESES OF THE RESEARCH: Many diagnostic techniques using genetic information are under developing now to create what is termed gPersonalized Medicineh - treatment designed for each tumor. However, important information is missing in the databases for detailed diagnostics and that is the information on the behavior of the gene products. The method developed in this proposal is expected to construct a database of changes in cell proteins behavior during various conditions including in response to chemotherapeutic drugs. These changes allowing cells to respond to extracellular stimuli is called signal transduction. I proposed a means to identify all the components that respond to activation of one signaling enzyme called delta PKC. Delta PKC activity is altered in tumor cells in response to extracellular stimuli that instruct it to divide faster and grow as well as to stimuli that tell it to die. Therefore, delta PKC interacts with other proteins that cause or inhibit tumor growth. Although it is clear that this information is required to tailor better treatment for cancer patients, so far very limited information is available on proteins with which delta PKC interacts during such signal transduction events. THE GENERAL METHODOLOGY: To analyze the intercellular signaling mechanism of breast cancer, I will focus on the protein-protein interactions involved in the tumor growth and tumor death. I plan to 'hijack' a mechanism that cells use normally to label proteins with a tag (called ubiquitin) in an irreversible way when these proteins need to be destructed (protein degradation). I plan to use such a tag, but design it so that it will bind on to proteins when they interacted with delta PKC. Using this tag, I will then purify the proteins of interest. The purified protein will be identified using Mass Spectrometry. All the experiments above will be carried out using human MCF-7 cultured breast cancer cell line, a cell line commonly used as a model of breast cancer. INNOVATIVE ELEMENTS OF THE PROJECT: The innovations of this proposal: 1) This is the first ubiquitin tagging system to be used to label proteins in living cells for a purpose other than their destruction. 2). Because the ubiquitin machinery is present in all cells, we can perform the analysis in any tumor type, in the normal cellular environment. 3) The method is novel in that it does not depend on long term association of delta PKC with other proteins. 4) The sponsorfs lab has already developed the delta PKC-specific inhibitor and activator reagents. Using these reagents, I can selectively manipulate delta PKC without affecting many other proteins that respond to extracellular stimuli. 5) The method can be quickly adapted to the study of other signaling events and thus help identify all the changes in proteins, or the PROTEOMICS of different tumor types.